Helicobacter pylori is a fastidious, microaerophilic organism strongly associated with peptic ulcers and gastric carcinoma. Growth requirements of this organism have only been partially defined, and isolation of H. pylori from in vivo sites and the environment remains challenging. H. pylori normally requires blood or serum for growth in vitro. Our preliminary experiments provide insights into growth and serum requirements and reveal that recovery of H. pylori from frozen stocks is significantly improved through the use of a partially defined medium rather than standard complex media. We hypothesize that the use of complex media inhibits recovery of dormant or oxidatively stressed organisms, precluding isolation of H. pylori from environmental sources. We propose to extend our preliminary studies by further defining growth requirements and identifying the component(s) of serum most responsible for growth augmentation. A transposon library of H. pylori mutants will be screened to identify clones which fail to grow in defined medium without serum. Since serum both augments growth and prolongs culture viability, genes identified in this screen are expected to provide clues regarding serum's function in H. pylori growth. These genes may also play a role in stationary phase survival of H. pylori and may impact virulence and transmission of the organism as well. The optimized medium will be used in conjunction with other measurements of viability to evaluate extragastric survival of H. pylori from sites such as the intestine, saliva, and feces. Results of these studies should extend the understanding of H. pylori growth requirements and the genes involved, particularly requirements relevant to recovery of the organism from various environmental conditions. This research will provide more useful media formulations for both clinical and research applications, and may also be applicable to the culture of other fastidious bacteria.